Isoprenoid-chained lipid β-XylOC16+4—A novel molecule for in meso membrane protein crystallization
Introduction
Membrane proteins are the main functional units of membranes and represent roughly one-third of the proteins encoded in the genome. In spite of the fact that approximately 70% of drugs have membrane proteins as their target, only about 1% of structures in the Protein Data Bank are membrane proteins. X-ray protein crystallography is one of the most powerful tools to determine protein structure and to provide the basis for understanding molecular mechanisms of protein function. However, high resolution X-ray diffraction studies require well ordered protein crystals, but unfortunately crystallization of membrane proteins is still a challenge. One of the most promising methods to overcome this challenge is the in meso crystallization approach, where the lipid cubic or sponge phase is used as a crystallization matrix. Recently, this method was demonstrated to be applicable to different membrane proteins including G protein-coupled receptors [1], membrane protein complexes [2], [3] and bacteriorhodopsin (bR) [4], which for a long time failed to be crystallized by the in surfo method. For the in meso crystallization 1-oleoyl-rac-glycerol (monoolein, MO) is generally used to create a bicontinuous lipid bilayer crystallization matrix.
Here we present our results of the successful use of a new lipid – 1-O-(3,7,11,15-tetramethylhexadecyl)-β-d-xyloside (β-XylOC16+4) – for bR in meso crystallization. Usually bR crystals obtained by in meso crystallization are perfectly twinned, which significantly complicates structural analysis and is probably one of the main reasons for controversial mechanisms of bR function [5]. Hexagonal plate bR crystals grown in the new lipid mesophase diffracted well and had zero or low twinning ratio. Due to the characteristic molecular shape of β-XylOC16+4 it was possible to identify the volume occupied by the host lipid in the crystal structure.
Section snippets
Synthesis and purification of β-XylOC16+4
β-XylOC16+4 was synthesized as described in the previous papers [6], [7], [8], [9], [10] and purified as in [11]. The β-anomeric purity of the sample checked by NMR, gas chromatography and TLC was at least 99.3%.
bR expression, purification and crystallization
Purple membranes were extracted from Halobacterium salinarium S9 [12]. bR was solubilized in n-octyl-β-d-glucoside (OG) and purified as described in detail in [13] and crystallized in the lipidic meso phase formed by β-XylOC16+4. The protein was mixed with lipid in the ratio 1 μl of 10
bR crystallization in β-XylOC16+4
There are a lot of compounds that can be screened to improve the number of successful membrane protein in meso crystallization trials such as lipids, detergents, small molecule additives and traditional crystallization reagents, etc. Using different classes of known detergents (with different combinations of both polar headgroups and hydrophobic chain lengths) it is possible to choose the detergent most suitable for each particular membrane protein target [21]. Unfortunately this is not the
Conclusions
The isoprenoid-chained lipid β-XylOC16+4 used as a host lipid for in meso crystallization provided high quality bR crystals diffracting to high resolution and further characterized by low twinning ratio. β-XylOC16+4 has a characteristic shape, which makes it clearly distinguishable in the membrane protein crystalline lattice, where it is shown to stabilize protein contacts. We suppose that β-XylOC16+4 may have a general application for in meso crystallization of a wide range of membrane
Acknowledgements
We are thankful to K.-E. Jaeger for the support of this work and C. Baeken for the help with bR production.
We would like to thank the ESRF and ESRF Structural Biology Group for the opportunity and help with data collection.
This work was financially supported in part by an NEDO International Joint Research Program (Subject: Molecular device for hydrogen production), the AIST Basic Research Program (Subject: Studies on bionanomaterials), the Targeted Proteins Research Program (TPRP) from Ministry
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